1. Equilibrate the system with at least 3 column volumes (CV) of
SEC running buffer and evaluate system performance with the
molecular weight calibrants.
2. We recommend (in particular if the column is new) to prime
the system with 700 μg of whole cell lysate (70% of the maximum column capacity) before the separation experiment.
3. Flush the system with at least 2 column volume between different injections.
4. Inject the concentrated SEC samples with the method
described above. An example of the UV traces and a standard
calibration curve is shown in Fig. 2.
5. Wash 96-Well Filter Plate with 200 μL of HPLC grade water.
6. Centrifuge at 1800 Â g for 30 min at room temperature.
7. Repeat steps 4 and 5 for a total of 2 washes.
8. Transfer 96-Well Filter Plates in the fraction collector to collect
the sample fractionated by the SEC (see Notes 11 and 12).
9. Monitor and evaluate the run quality using the chromatogram
displayed in the software.
log10(DA)
normalized mABS
fraction
volume (mL)
(1)
(2)
0
2 0
4 0
6 0
6 7 0
k D
a 3 0 0
k D
a
1 5 0
k D
a 4 4
k D
a 1 7
k D
a
0
100
200
300
400
15
5 6 7 8 9 10 11 12
OD260
OD280
0
2
4
6
log10(MW) = 6.92 - 0.0571 Fr, R² = 0.98
6 7 0
k D
a 3 0 0
k D
a
1 5 0
k D
a 4 4
k D
a 1 7
k D
a
Fig. 2 Representative whole cell lysate OD280 UV trace on a Yarra SEC4000 and corresponding standard
calibration curve. (1) OD280/260 profile of a native Jurkat cell lysate ($1 mg injection amount) fractionated
into 60 fractions each with 125 μL on a Yarra SEC4000 (separation conditions as described under Subheading
3.4). The UV traces show the characteristic peaks of a full lysate, i.e., elution of unfractionated large
macromolecular assemblies in the void volume (4.75–6 mL) and NP-40% detergent peak at 12.25 mL. This
setup allows to separate protein complexes from around 1.5 MDa to 10 kDa and thus cover the vast majority of
macromolecular assemblies in a human sample. Molecular weight standard proteins are marked in vertical
dotted blue lines, allow to estimate protein complex weight. (2) To monitor separation performance, a
molecular weight standard mixture (MW standard mixture, Phenomenex, AL0-3042) was injected on the
SEC-column before and after the whole cell lysate sample. The log10 MW of standards in Dalton and the
fraction number, respectively, volume are used to estimate MW calibration curve
System-Wide Profiling of Protein Complexes Via Size Exclusion. . .
279
SEC running buffer and evaluate system performance with the
molecular weight calibrants.
2. We recommend (in particular if the column is new) to prime
the system with 700 μg of whole cell lysate (70% of the maximum column capacity) before the separation experiment.
3. Flush the system with at least 2 column volume between different injections.
4. Inject the concentrated SEC samples with the method
described above. An example of the UV traces and a standard
calibration curve is shown in Fig. 2.
5. Wash 96-Well Filter Plate with 200 μL of HPLC grade water.
6. Centrifuge at 1800 Â g for 30 min at room temperature.
7. Repeat steps 4 and 5 for a total of 2 washes.
8. Transfer 96-Well Filter Plates in the fraction collector to collect
the sample fractionated by the SEC (see Notes 11 and 12).
9. Monitor and evaluate the run quality using the chromatogram
displayed in the software.
log10(DA)
normalized mABS
fraction
volume (mL)
(1)
(2)
0
2 0
4 0
6 0
6 7 0
k D
a 3 0 0
k D
a
1 5 0
k D
a 4 4
k D
a 1 7
k D
a
0
100
200
300
400
15
5 6 7 8 9 10 11 12
OD260
OD280
0
2
4
6
log10(MW) = 6.92 - 0.0571 Fr, R² = 0.98
6 7 0
k D
a 3 0 0
k D
a
1 5 0
k D
a 4 4
k D
a 1 7
k D
a
Fig. 2 Representative whole cell lysate OD280 UV trace on a Yarra SEC4000 and corresponding standard
calibration curve. (1) OD280/260 profile of a native Jurkat cell lysate ($1 mg injection amount) fractionated
into 60 fractions each with 125 μL on a Yarra SEC4000 (separation conditions as described under Subheading
3.4). The UV traces show the characteristic peaks of a full lysate, i.e., elution of unfractionated large
macromolecular assemblies in the void volume (4.75–6 mL) and NP-40% detergent peak at 12.25 mL. This
setup allows to separate protein complexes from around 1.5 MDa to 10 kDa and thus cover the vast majority of
macromolecular assemblies in a human sample. Molecular weight standard proteins are marked in vertical
dotted blue lines, allow to estimate protein complex weight. (2) To monitor separation performance, a
molecular weight standard mixture (MW standard mixture, Phenomenex, AL0-3042) was injected on the
SEC-column before and after the whole cell lysate sample. The log10 MW of standards in Dalton and the
fraction number, respectively, volume are used to estimate MW calibration curve
System-Wide Profiling of Protein Complexes Via Size Exclusion. . .
279